Seasonal and Daily Activity Patterns of Mosquito (Diptera: Culicidae) Vectors of Pathogens in Northeastern Italy.

The seasonal and daily activity of mosquito vectors of pathogens affecting animals and humans were studied in northeastern Italy at a site within the Po River Delta Park. A CDC-CO2 trap and a gravid trap were operated at 2-h intervals for 24 h every 15 d from May to October 2010. Overall, 5,788 mosquitoes comprising six species were collected, namely Culex pipiens L. (75.1% of total), Aedes caspius (Pallas) (15.2%), Aedes vexans (Meigen) (6.9%), Anopheles maculipennis s.l. Meigen (2.6%), Culiseta annulata (Schrank) (0.2%), and Culex modestus Ficalbi (<0.1%). The relative abundance of these species increased from May until the beginning of July and then decreased, disappearing at the beginning of October. The diel host-seeking patterns and oviposition site-seeking patterns were species specific and were differentially affected by the ecological variables recorded at the day and hour of mosquito collection or two weeks before collection. Knowledge of the seasonal and daily host-seeking patterns of mosquitoes highlights the time periods of the day and the seasons of potential exposure for animals and humans to mosquito-borne pathogens, therefore delineating the best time for the application of preventive measures. Furthermore, knowledge of the oviposition site-seeking activity of the mosquitoes optimizes the capture of gravid females, thereby enhancing the likelihood of detecting pathogens.

Respiratory nematodes in cat populations of Italy.

The occurrence of common respiratory parasites of domestic cats (the metastrongyloid "cat lungworm" Aelurostrongylus abstrusus and the trichuroid Capillaria aerophila) and of neglected respiratory nematodes of felids (Troglostrongylus brevior, Angiostrongylus chabaudi and Oslerus rostratus) was here evaluated in two and three geographical sites of Northern and Central Italy, respectively. In 2014-2015, individual fecal samples of 868 domestic cats were examined microscopically and genetically, and epidemiological data related to parasitic infections were evaluated as possible risk factors by binary logistic regression models. The most common parasite was A. abstrusus in both mono- and poli-specific infections, followed by T. brevior and C. aerophila, while cats scored negative for other parasites. Cats positive for A. abstrusus (1.9-17 % infection rate) and C. aerophila (0.9-4.8 % infection rate) were found in all examined sites, while cats scored positive for T. brevior (1-14.3 % infection rate) in four sites. Also, T. brevior was here found for the first time in a domestic cat from a mountainous area of Northern Italy. The occurrence of lungworms was statistically related to the presence of respiratory signs and more significant in cats with mixed infection by other lungworms and/or intestinal parasites. Cats living in site C of Central Italy resulted statistically more at risk of infection for lungworms than cats living in the other study sites, while animals ageing less than 1 year were at more risk for troglostrongylosis. Finally, the presence of lungworms was more significant in cats with mixed infection by other lungworms and/or intestinal parasites. These results are discussed under epidemiological and clinical points of views.

First Detection of West Nile Virus Lineage 2 in Mosquitoes in Switzerland, 2022.

West Nile virus (WNV) is one of the most widespread flaviviruses in the world, and in recent years, it has been frequently present in many Mediterranean and Eastern European countries. A combination of different conditions, such as a favourable climate and higher seasonal average temperatures, probably allowed its introduction and spread to new territories. In Switzerland, autochthonous cases of WNV have never been reported, and the virus was not detected in mosquito vectors until 2022, despite an entomological surveillance in place in Canton Ticino, southern Switzerland, since 2010. In 2022, 12 sites were monitored from July to October, using BOX gravid mosquito traps coupled with honey-baited FTA cards. For the first time, we could detect the presence of WNV in FTA cards and mosquitoes in 8 out of the 12 sampling sites monitored, indicating an unexpectedly widespread circulation of the virus throughout the territory. Positive findings were recorded from the beginning of August until mid-October 2022, and whole genome sequencing analysis identified a lineage 2 virus closely related to strains circulating in Northern Italy. The entomological surveillance has proved useful in identifying viral circulation in advance of possible cases of WNV infection in humans or horses.

Exposure to vector-borne pathogens in candidate blood donor and free-roaming dogs of northeast Italy.

BACKGROUND: Many vector-borne pathogens including viruses, bacteria, protozoa and nematodes occur in northeast Italy, representing a potential threat to animal and human populations. Little information is available on the circulation of the above vector-borne pathogens in dogs. This work aims to (i) assess exposure to and circulation of pathogens transmitted to dogs in northeast Italy by ticks, sandflies, and mosquitoes, and (ii) drive blood donor screening at the newly established canine blood bank of the Istituto Zooprofilattico Sperimentale delle Venezie. METHODS: Blood samples from 150 privately-owned canine candidate blood donors and 338 free-roaming dogs were screened by serology (IFA for Leishmania infantum, Ehrlichia canis, Anaplasma phagocythophilum, Babesia canis, Rickettsia conorii, R. rickettsii), microscopic blood smear examination, and blood filtration for Dirofilaria spp. All candidate donors and seropositive free-roaming dogs were tested by PCR for L. infantum, E. canis, A. phagocythophilum, Babesia/Theileria and Rickettsia spp. The dogs had no clinical signs at the time of sampling. RESULTS: Overall, 40 candidate donors (26.7 %) and 108 free-roaming dogs (32 %) were seroreactive to at least one vector-borne pathogen. Seroprevalence in candidate donors vs free-roaming dogs was: Leishmania infantum 6.7 vs 7.1 %; Anaplasma phagocytophilum 4.7 vs 3.3 %; Babesia canis 1.3 vs 2.7 %; Ehrlichia canis none vs 0.9 %; Rickettsia conorii 16 vs 21.3 % and R. rickettsii 11 vs 14.3 %. Seroreactivity to R. rickettsii, which is not reported in Italy, is likely a cross-reaction with other rickettsiae. Filariae, as Dirofilaria immitis (n = 19) and D. repens (n = 2), were identified in free-roaming dogs only. No significant differences were observed between candidate donors and free-roaming dogs either in the overall seroprevalence of vector-borne pathogens or for each individual pathogen. All PCRs and smears performed on blood were negative. CONCLUSIONS: This study demonstrated that dogs are considerably exposed to vector-borne pathogens in northeast Italy. Although the dog owners reported regularly using ectoparasiticides against fleas and ticks, their dogs had similar exposure to vector-borne pathogens as free-roaming dogs. This prompts the need to improve owner education on the use of insecticidal and repellent compounds in order to reduce the risk of arthropod bites and exposure to vector-borne pathogens. Based on the absence of pathogens circulating in the blood of healthy dogs, the risk of transmission of these pathogens by blood transfusion seems to be low, depending also on the sensitivity of the tests used for screening.

First report outside Eastern Europe of West Nile virus lineage 2 related to the Volgograd 2007 strain, northeastern Italy, 2014.

BACKGROUND: West Nile virus (WNV) is a Flavivirus transmitted to vertebrate hosts by mosquitoes, maintained in nature through an enzootic bird-mosquito cycle. In Europe the virus became of major public health and veterinary concern in the 1990s. In Italy, WNV re-emerged in 2008, ten years after the previous outbreak and is currently endemic in many areas of the country. In particular, the northeastern part of Italy experience continuous viral circulation, with human outbreaks caused by different genovariants of WNV lineage 1, Western-European and Mediterranean subcluster, and WNV lineage 2, Hungarian clade. Alongside the WNV National Surveillance Program that has been in place since 2002, regional surveillance plans were implemented after 2008 targeting mosquitoes, animals and humans. FINDINGS: In July and September 2014, West Nile virus lineage 2 was detected in pools of Culex pipiens s.l. mosquitoes from northeastern Italy. Whole genome sequencing and phylogenetic analysis of two representative samples identified the presence of WNV lineage 2 related to the Volgograd 2007 strain (99.3 % nucleotide sequence identity), in addition to WNV lineage 2 Hungarian clade. CONCLUSIONS: This is the first evidence of the circulation of a WNV lineage 2 strain closely related to the Volgograd 2007 outside Eastern Europe, where it has caused large human outbreaks. This strain may pose a new threat to animal and human health in Italy.

West Nile Virus Surveillance in 2013 via Mosquito Screening in Northern Italy and the Influence of Weather on Virus Circulation.

West Nile virus (WNV) is a recently re-emerged health problem in Europe. In Italy, an increasing number of outbreaks of West Nile disease, with occurrences of human cases, have been reported since 2008. This is particularly true in northern Italy, where entomological surveillance systems have been implemented at a regional level. The aim of this study was to use, for the first time, all the entomological data collected in the five regions undergoing surveillance for WNV in northern Italy to characterize the viral circulation (at a spatial and temporal scale), identify potential mosquito vectors, and specify relationships between virus circulation and meteorological conditions. In 2013, 286 sites covering the entire Pianura Padana area were monitored. A total of 757,461 mosquitoes were sampled. Of these, 562,079 were tested by real-time PCR in 9,268 pools, of which 180 (1.9%) were positive for WNV. The largest part of the detected WNV sequences belonged to lineage II, demonstrating that, unlike those in the past, the 2013 outbreak was mainly sustained by this WNV lineage. This surveillance also detected the Usutu virus, a WNV-related flavivirus, in 241 (2.6%) pools. The WNV surveillance systems precisely identified the area affected by the virus and detected the viral circulation approximately two weeks before the occurrence of onset of human cases. Ninety percent of the sampled mosquitoes were Culex pipiens, and 178/180 WNV-positive pools were composed of only this species, suggesting this mosquito is the main WNV vector in northern Italy. A significantly higher abundance of the vector was recorded in the WNV circulation area, which was characterized by warmer and less rainy conditions and greater evapotranspiration compared to the rest of the Pianura Padana, suggesting that areas exposed to these conditions are more suitable for WNV circulation. This observation highlights warmer and less rainy conditions as factors able to enhance WNV circulation and cause virus spillover outside the sylvatic cycle.

Retrospective and spatial analysis tools for integrated surveillance of cystic echinococcosis and bovine cysticercosis in hypo-endemic areas.

Cystic echinococcosis (CE) and bovine cysticercosis (BC) are two important parasitic zoonoses, whose prevalence varies among European countries. Few data are available on prevalence and geographic distribution of these two diseases in Veneto region in North-Eastern Italy, where they are generally perceived as minor public health problems. Available data from regional farms on cattle positive to CE and BC and slaughtered in the period 2006-2010 were analysed by spatial scan statistic using a Bernoulli probablility model. Out of 576 bovines testing positive to CE, 467 were found to be autochthonous cases. Three significant CE clusters were identified, the most likely one (P < 0.0001) located in the eastern part of the Veneto region. As for BC, two clusters were identified from 148 animals resting positive, 91 which were autochthonous. An epidemiological survey was conducted and the most likely CE cluster was centered, collecting faecal samples from 28 dogs living in the farms of the area. Out of five animals (all shepherd dogs) found positive for taenid eggs by copromicroscopy, one was confirmed positive for Echinoccus granulosus by means of polymerase chain reaction. The study demonstrates the usefulness of integration of slaughterhouse data and geographical coordinates of farms involved for effective surveillance of CE and BC. The reliability of the spatial analysis in the identification of clusters of EC cases was confirmed by the finding of one dog positive for E. granulosus.

Ticks infesting humans in Italy and associated pathogens.

BACKGROUND: Ticks may transmit a large variety of pathogens, which cause illnesses in animals and humans, commonly referred to as to tick-borne diseases (TBDs). The incidence of human TBDs in Italy is underestimated because of poor surveillance and the scant amount of studies available. METHODS: Samples (n = 561) were collected from humans in four main geographical areas of Italy (i.e., northwestern, northeastern, southern Italy, and Sicily), which represent a variety of environments. After being morphologically identified, ticks were molecularly tested with selected protocols for the presence of pathogens of the genera Rickettsia, Babesia, Theileria, Candidatus Neoehrlichia mikurensis, Borrelia and Anaplasma. RESULTS: Ticks belonged to 16 species of the genera Argas, Dermacentor, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus, with Ixodes ricinus (59.5%) being the species most frequently retrieved, followed by Rhipicephalus sanguineus sensu lato (21.4%). Nymphs were the life stage most frequently retrieved (41%), followed by adult females (34.6%). The overall positivity to any pathogen detected was 18%. Detected microorganisms were Rickettsia spp. (17.0%), Anaplasma phagocytophilum (0.8%), Borrelia afzelii (0.5%), Borrelia valaisiana (0.3%), C. N. mikurensis (0.5%) and Babesia venatorum (0.6%). CONCLUSIONS: Results indicate that people living in the Italian peninsula are at risk of being bitten by different tick species, which may transmit a plethora of TBD causing pathogens and that co-infections may also occur.

Risk of canine and human exposure to Dirofilaria immitis infected mosquitoes in endemic areas of Italy.

BACKGROUND: The occurrence of infections by Dirofilaria immitis in canine and human populations depends on several factors linked to both the definitive and intermediate hosts. Little data are available on the risk of human and dog exposure to D. immitis in endemic areas. Data collected on dog- and human-bait traps in endemic areas of north-eastern Italy were used to estimate the likelihood of a receptive host coming into contact with an infected vector. METHODS: From 1997 to 1999, mosquitoes were collected from three sampling sites of north-eastern Italy on D. immitis microfilaraemic dogs and on human baits. The bite/night/host rates were determined based on the number of feeding and probing mosquitoes on dogs and humans, respectively. The survival/mortality rates of different species of mosquitoes following the blood meal, and the rate of natural Dirofilaria infection in unfed specimens were estimated. The risk of exposure of dogs and humans to infected mosquito species was determined by combining the bite/host/night and the mosquito infection rates. RESULTS: A total of 1,165 mosquitoes were collected on human (n=815) and dog (n=350) baits with varying species composition (i.e., Culex pipiens, 87.3% and Ochlerotatus caspius, 11.6%). Overall, dogs were more attractive to Cx pipiens than humans (feeding rate 70.2% vs probing rate 25.9%). The highest bite/night/host rate was 84.0 for dogs and 26.5 for humans. Cx pipiens displayed a mortality rate of 76.3% within 13 days and Oc. caspius of 100% within two days following the infective blood meal. In addition, D. immitis DNA was detected in unfed Cx pipiens (infection rate of 0.26%-2.07%). The infection rate adjusted for mosquito mortality was 0.38%. Based on data collected, the contact between an infected mosquito and a host can occur as often as every four nights for D. immitis infected-mosquitoes in dogs and within two weeks for humans. CONCLUSIONS: Cx pipiens was confirmed as the most efficient natural vector of D. immitis in the studied area. In endemic areas, the risk of transmission can be very high for dogs and relevant for humans. Despite the increased awareness of veterinarians and owners on canine dirofilarioses, dogs from rural areas still maintain the natural life cycle of Dirofilaria spp., therefore acting as a source of infection to humans through vector bites.

Further evidence of lineage 2 West Nile Virus in Culex pipiens of North-Eastern Italy.

West Nile Virus lineage 1 (WNV lin1) emerged in North-Eastern Italy in 2008 and, since then, it has been detected in animals, humans and mosquitoes. Three years later, in the same area, a lineage 2 (lin2) strain of WNV was found in birds and vectors. On August the 21st, during the 2012 WNV entomological surveillance plan, a WNV lin2 strain was detected by RT-PCR in a pool of Culex pipiens mosquitoes captured in Veneto region. According to the alignment of the partial sequences of the NS5 and NS3 genes, no differences between this Italian lineage 2 strain and the Nea Santa-Greece-2010 WNV isolate (Gr-10) were observed. Similarly to the Gr-10 strain, the putative NS3 amino acid sequences of the Italian strain showed proline in position 249 instead of histidine (H249P). Although proline in position 249 has been suggested to increase the virulence of WNV strains, neither human nor veterinary cases associated to this strain have been reported in the region. A prompt mosquito disinfestation was organized to avoid the spread of this potential threatening virus. The simultaneous circulation of both WNV lineage 1 and 2 confirms North-Eastern Italy as a high risk area for WNV emergence and highlights the need for a continuous surveillance.

Distribution and habitat characterization of the recently introduced invasive mosquito Aedes koreicus [Hulecoeteomyia koreica], a new potential vector and pest in north-eastern Italy.

BACKGROUND: The container breeding species belonging to the genus Aedes (Meigen) are frequently recorded out of their place of origin. Invasive Aedes species are proven or potential vectors of important Arboviruses and their establishment in new areas pose a threat for human and animal health. A new species of exotic mosquito was recorded in 2011 in north-eastern Italy: Aedes (Finlaya) koreicus [Hulecoeteomyia koreica]. The aim of this study was to characterize the biology, the environment and the current distribution of this mosquito in north-eastern Italy. Morphological details useful to discriminate this species from other invasive Aedes mosquitoes are also given (see Additional files). METHODS: All possible breeding sites for larval development were monitored. In addition, ovitraps and traps for adults were used to collect eggs and adults. The mosquitoes (larvae and adults) were identified morphologically and molecularly. Environmental data and climatic variables during the period of mosquito activity (from April to October) were considered. RESULTS: Aedes koreicus was found in 37 municipalities (39.4%) and was detected in 40.2% of places and in 37.3% of larval habitats monitored, in a range of altitude from 173 to 1250 m.a.s.l.. Garden centres were the most common locations (66.7%), followed by streets/squares (57.1%), private gardens (46.4%) and cemeteries (21.1%) (p < 0.01). The main larval habitats were catch basins (48.5%) and artificial water containers (41.8%). As for Aedes albopictus [Stegomyia albopicta], ovitraps were attractive for adult females resulting in the higher rate of positivity (15/21; 71.4%) among breeding sites. The period of Ae. koreicus activity ranged from March 29 to October 29. CONCLUSION: The species is clearly established in the area and is now overlapping with other vectors such as Ae. albopictus and colonizing areas over 800 m.a.s.l, not yet or sporadically reached by the tiger mosquito. The data collected are essential to assess the risk of colonization of other parts of Italy and Europe, as well as the risk of spreading of pathogens transmitted. These findings stress the importance of implementing entomological surveillance for early detection of invasive species, which is necessary for eradication or limitation of its further spread.

New incursions of West Nile virus lineage 2 in Italy in 2013: the value of the entomological surveillance as early warning system.

West Nile virus (WNV) is one of the most serious public health threats that Europe and the Mediterranean countries are currently facing. In Italy, WNV emerged in 1998 and has been circulating since 2008. To tackle its continuous incursions, Italian national and regional institutions set up a surveillance program, which includes the serological screening of sentinel horses, sentinel-chickens and backyard poultry flocks and the surveillance on all equine neurological cases, resident captured and wild dead birds, and vectors. This communication aims to assess the importance of the entomological surveillance program as an early warning system for WNV circulation. In the province of Modena, the circulation of WNV lineage 2 strains was first detected in pools of Culex pipiens on July the 3rd, 42 days prior to the onset of the first 2013 human WNV neuroinvasive case reported in the same province. Similarly in Veneto, WNV was first detected on July 3rd in a pool of Cx. pipiens collected in the province of Venezia. The first human neuroinvasive case in this region occurred in the Rovigo province on July the 24th, seven days after the detection of WNV lineage 2 in a mosquito pool collected in the same province. Up to the end of July 2013, WNV circulation was further detected in several other pools of Cx. pipiens mosquitoes collected in Emilia-Romagna, Veneto and Lombardia. According to the NS3 partial sequence alignments including all recent European and Italian Lineage 2 strains, the new circulating WNV lineage 2 strains share high nt homology with the Hungarian and with the previous lineage 2 strains isolated in Veneto and Sardegna in 2011 and 2012. These data provide a clear and practical demonstration of the relevance of a reliable entomological surveillance program to early detect WNV in Italy.

Occurrence and identification of risk areas of Ixodes ricinus-borne pathogens: a cost-effectiveness analysis in north-eastern Italy.

BACKGROUND: Ixodes ricinus, a competent vector of several pathogens, is the tick species most frequently reported to bite humans in Europe. The majority of human cases of Lyme borreliosis (LB) and tick-borne encephalitis (TBE) occur in the north-eastern region of Italy. The aims of this study were to detect the occurrence of endemic and emergent pathogens in north-eastern Italy using adult tick screening, and to identify areas at risk of pathogen transmission. Based on our results, different strategies for tick collection and pathogen screening and their relative costs were evaluated and discussed. METHODS: From 2006 to 2008 adult ticks were collected in 31 sites and molecularly screened for the detection of pathogens previously reported in the same area (i.e., LB agents, TBE virus, Anaplasma phagocytophilum, Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis"). Based on the results of this survey, three sampling strategies were evaluated a-posteriori, and the impact of each strategy on the final results and the overall cost reductions were analyzed. The strategies were as follows: tick collection throughout the year and testing of female ticks only (strategy A); collection from April to June and testing of all adult ticks (strategy B); collection from April to June and testing of female ticks only (strategy C). RESULTS: Eleven pathogens were detected in 77 out of 193 ticks collected in 14 sites. The most common microorganisms detected were Borrelia burgdorferi sensu lato (17.6%), Rickettsia helvetica (13.1%), and "Ca. N. mikurensis" (10.5%). Within the B. burgdorferi complex, four genotypes (i.e., B. valaisiana, B. garinii, B. afzelii, and B. burgdorferi sensu stricto) were found. Less prevalent pathogens included R. monacensis (3.7%), TBE virus (2.1%), A. phagocytophilum (1.5%), Bartonella spp. (1%), and Babesia EU1 (0.5%). Co-infections by more than one pathogen were diagnosed in 22% of infected ticks. The prevalences of infection assessed using the three alternative strategies were in accordance with the initial results, with 13, 11, and 10 out of 14 sites showing occurrence of at least one pathogen, respectively. The strategies A, B, and C proposed herein would allow to reduce the original costs of sampling and laboratory analyses by one third, half, and two thirds, respectively. Strategy B was demonstrated to represent the most cost-effective choice, offering a substantial reduction of costs, as well as reliable results. CONCLUSIONS: Monitoring of tick-borne diseases is expensive, particularly in areas where several zoonotic pathogens co-occur. Cost-effectiveness studies can support the choice of the best monitoring strategy, which should take into account the ecology of the area under investigation, as well as the available budget.